Microfluidic assisted low-temperature and speedy synthesis of TiO2/ZnO/GOx with bio/photo active cites for amoxicillin degradation

For the first time, a bio-photo-catalyst is synthesized in a microfluidic platform. The microchannel, which is wall-coated by in situ synthesized bio-photo-catalyst is used as an opto-fluidic reactor for amoxicillin degradation. Analyses including SEM, XRD, FTIR, Raman, UV–Vis spectra, and DLS have been used to characterize samples. The structure and morphology of TiO2 in microfluidic assisted synthesis are studied at 70–120 °C. The results show that both single-crystalline anatase sample and two-phase samples of anatase and rutile can be attained. According to SEM images, the smallest size and the narrowest particle size distribution (0.86 nm \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \hspace{0.17em}0.14$$\end{document}±0.14) is achieved by synthesis at 70 °C. Elemental mapping of Ti shows a uniform coating layer on inner walls. Raman signals besides the primary amines in FTIR results show the biological activity of the cross-linked Glucose oxidase (GOx), which is aimed for situ generation of H2O2. FTIR comparison of bulk and spiral microfluidic synthesized ZnO indicates identical bonds. SEM-coupled with performance experimentation reveal that by regulating the flowrate of spiral micromixer for ZnCl2 at 25 µl/min and NaOH at 50 µl/min, the narrowest size distribution and best the bio-photo-catalytic performance of ZnO nanoparticles is observed.


Material and methods
Materials. Titanium (III) chloride (> 15% TiCl 3 basis, 5-10% free acid as HCl, Sigma-Aldrich) was used in the synthesis of TiO 2 . For ZnO synthesis, zinc chloride (> 98%, Merck) and NaOH (pellets > 97%, Merck) have been used. For rinsing the samples, isopropyl alcohol (> 99.8%, Merck) and double distilled water were used, the latter been prepared in the Research Laboratory at the chemical engineering Department of Amirkabir University of Technology. The Glutardialdehyde solution (25%, Merck), and GOx is gifted from Bonda Group Development. Glucose monohydrates has been employed as the substrate for GOx. The target water contaminant is Amoxicillin trihydrate, which is received from Afa Chimi. Sodium 1, 2-naphthoquinone-4-sulfonate (> 97%, Merck) acts as the chemical derivative chromogenic reagent, K 2 HPO 4 (> 98%, Merck), and KH 2 PO 4 (> 99.5%, Merck) for adjusting pH are used in the visible spectroscopic detection of amoxicillin. PDMS (Sylgard 184 Silicon Elastomer Kit, Dow Corning) is used in the microfabrication of the spiral chip.
Methods. Microfluidic synthesis of TiO 2 . According to Fig. 1a, TiO 2 is synthesized and then ZnO nanoparticles and glucose oxidase enzyme have been immobilized on its surface in two consecutive steps. For this purpose, a solution of TiCl 3 precursor in water with a concentration of 0.1 v/v at a speed of 1 mm/min, at several temperatures including 60, 70, 80, 90, 100, 120 °C was injected into the capillary tube. These injection cycles have been repeated up to 4 times to devote a total of 100 min for synthesis. To make the coated layer more annealed and durable, the capillary tubes were transferred to an oven and heated at 140 °C for 1 h, and the output solution was dried at the same temperature and some powders were obtained. In Fig. 1b, the real TiO 2 sample images are also given to highlight the color change owing to the synthesis temperature.
Microfabrication of ZnO synthesis chip. During ZnO synthesis, ZnCl 2 to NaOH streams with concentrations of 30 mM and 50 mM should be mixed in a spiral micromixer, with cross section of 200 µm × 100 µm. The chip is made of PDMS and created on a lamella with plasma bonding, which includes 4 rings with a distance of 2 mm and two Y-shaped inputs and one output. The molding steps are as follows: 1 Combining Silgard 184 with a curing agent at a ratio of 10 to 1 weight and completely stirring. 2 Debubble with a desiccator for 30 min 3 Pour the PDMS mixture into the mold and bake on a hot plate at 90 degrees for 15 min 4 Open and separate the PDMS from the wafer and cut it to the desired size and create the input and output with a 1 mm tissue cutting punch. 5 Cleaning lamellar glass pieces with suitable dimensions with acetone, propanol, and deionized water 6 Drying the glass with clean and dry air 7 Cleaning PDMS with adhesive tape to remove any particles attached to it 8 Placing PDMS and lamellas in the plasma bonding chamber 9 Exposure to plasma with oxygen plasma at a pressure of 8 e-1 m bar, with a power of 40 watts for 1 min 10 Remove the parts and paste the PDMS and glass to each other and heat on a hot plate at 90 degrees for 5 min. Characterization. The characterization tests include XRD, SEM, DLS, FTIR, UV-Vis and RAMAN spectroscopy. The crystalline phase identification, and crystallite size of TiO 2 powders, which are collected from the effluents of the capillaries were characterized by X-ray diffraction (XRD) using DIFFRACTOMETER of inel CO. EQUINOX3000 model with Cu Ka radiation: 1.54190 A at 40 kV and 30 mA. The XRD patterns were collected from 5 to 90° in 2ɵ range at a scan rate of 0.032°/s. The crystallite size was calculated from peak broadening using the Sherrer formula. The phases of samples have been detected by means of XPert HighScore Plus software. The morphology of the wall coated TiO 2 synthesized at temperatures in the range of 70-120 °C, and ZnO nanoparticles, which have been synthesized at different flow rates within a spiral micromixer, were investigated using a scanning electron microscope of Philips Company, XL300 model at the acceleration voltage of 25 kV.
To compare the functional groups of ZnO powder, which is obtained from bulk and microfluidic systems, and to confirm the presence of bio and photo-active cites of the biophotocatalyst, Fourier transform infrared (FTIR) of ZnO, GOx, and TiO 2 /ZnO/GOx have been collected using a Perkin-Elmer Spectrum, Frontier model, Version 10.03.06 (Perkin-Elmer Instruments, Norwalk, CT, USA) in the range of 400-4000 cm −1 . Raman signals from the biophotocatalyst have been collected using a Raman Microspectrometer in the range of 400-3200 cm −1 by a green laser operating at 532 nm with an incident power of 10 mW with a resolution of 16 cm −1 . DLS (Dynamic Light Scattering) of Cordouan Tech company, VASCO2 model has been utilized to estimate the impact of synthesis temperature on the size of TiO 2 powders. Analytical methods. After the bio-photo-catalysis, DR 3900 spectrophotometer of HACH CO. is used to determine amoxicillin degradation efficiency by a colorimetric method. To 8 mL of amoxicillin, 2.0 mL of 0.2% sodium 1, 2-naphthoquinone-4-sulfonate and 2.0 mL K 2 HPO 4 -KH 2 PO 4 buffer solution of pH 8.50 are sequentially added. Then, the mixture undergoes room temperature shaking at 150 rpm for 50 min. The blank solution prepared with the same procedure and reagent composition, but no amoxicillin. Finally, visible spectroscopic detection of amoxicillin will be possible at 468 nm, which is much more facile and cost-effective than high-pressure liquid chromatography (HPLC) and more accurate than UV spectroscopy. The method is adopted www.nature.com/scientificreports/ from 14 with some modifications. The amoxicillin degradation efficiency has been calculated using the following equation, which is derived from Beer-Lambert law.

Results and discussions
Cross-sectional view of TiO 2 coated microchannels. To observe how temperature influences the thickness of the synthesized TiO 2 layer, SEM snapshots of capillaries cross-sections at different scales including 500, 100, 50 and 10 µm and various synthesis temperatures from 70 to 120 °C have been taken. The yellow arrows represent the thickness of the layer. According to Fig. 2, at 70 and 80 °C, the TiO 2 layer is more uniform and the effect of temperature on the layer thickness is incremental. At 90 °C, particles of different sizes are formed, which causes the thickness of the layer to increase with a greater slope. At 100 °C, due to the initiation of more nucleation, more particles are formed with smaller sizes. At 120 °C, particles with more regular shapes and relatively close sizes are formed, which can be due to the balanced longitudinal and transverse growth of the TiO 2 structure. TiO 2 growth rate and histogram. According to Fig. 2 and with aid of image j software, the following histogram ( Fig. 3a) is attained for TiO 2 layer thickness, which shows the effect of temperature in the range of 70-120 °C is not inevitably ascending. In other words, from 70 to 90 °C, the layer thickness and its standard deviation increase. From 90 to 120 °C, the layer thickness decreases. At 90 °C, due to secondary nucleation, it is a turning point in microfluidic synthesis. Such behavior was observed in the bulk system at 180 °C. The ideal synthesis temperature can be 70 °C. First, TiO 2 size is smaller, which is larger according to 15 corresponding to the specific surface area. Second, size distribution is the narrowest. Figure 3b shows a graph of TiO 2 layer thickness increase and longitudinal growth. Growth rate ((L T -L 70 )/∆T) is considered based on the rate of the average thickness increase at each temperature relative to the average layer thickness at 70 °C divided by its temperature difference with 70 °C.
Due to the increase in temperature from 70 to 80 °C, the layer thickness increases from 860 to 2.61 mm. When the synthesis temperature increases from 70 to 90 °C, the layer thickness reaches 15.92 µm, and the growth rate increases more than 4 times. Once the synthesis temperature increases from 70 to 100 °C, the layer thickness reaches 14.77 µm, and the growth rate increases more than 2.5 times. With the synthesis temperature increasing from 70 to 120 °C, the layer thickness reaches 11.82 µm, and the growth rate increases more than 1.25 times. Fig. 4, the synthesis at 70 °C resulted in the production of 50 nm particles that are homogeneously dispersed on the surface. A few nanoclusters with a maximum diameter of 700 nm are also witnessed, but they are much smaller in size and frequency compared to the ones detected at higher synthesis temperatures. As the temperature increases, a number of secondary nuclei can be seen, which peaks at 90 °C. At 80 °C, the presence of several particles about 5 microns in length is seen, indicating an increase in the particle size distribution range. At 90 °C, the number of larger particles is greater. At 100 °C more particles are formed with smaller sizes. At 120 °C high surface porosity and particle growth can be seen. EDX results confirm the uniformity of the coating and the presence of titanium dioxide. Hurtado et al. 2016 proved that the photonic efficiency is two orders of magnitude higher in the coated capillary reactor than in the slurry stirred reactor (STR), and it is twice slurry capillary 16 . It should be noted that an aqueous solution is forced through a micro-packed bed capillary, it will tend to circumnavigate the beads instead of passing through them, which affects the yield of the process 17 .

Surface study of the inner wall microchannel. According to the SEM images shown in
How synthesis cycles affects TiO 2 size. To investigate the effect of synthesis time on morphology, following the synthesis procedures, the only variable was the synthesis time. Therefore, the precursor solutions are injected into the capillaries for three and five consecutive cycles at 80 °C. A total of 75 and 125 min were devoted to synthesis, and then the capillaries are transferred to a conventional oven and heated at 140 °C for 1 h to make the coated TiO 2 layer durable. As shown in Fig. 5, the particle size increases with increasing synthesis time, as well as the cracks on the surface.
The histograms indicates that synthesis time of 75 min leads to TiO 2 mean size of 7.11 nm with 2.04 nm standard deviation. Once synthesis time increases to its 1.67fold, both the particle size and size distribution proliferates more than two times.
Crystallographic study of TiO 2 powder. The XRD spectrum of samples synthesized at different temperatures in the range of 60-120 °C is given in Fig. 6. It is noted that these samples are collected from the effluents of the capillaries. With synthesis temperature of 60 °C, two anatase peaks of (101) and (200) planes appear. As the temperature rises to 80 °C, the sum of peaks proliferates. where "I" represents the maximum peak intensity, A represents anatase and R denotes rutile. The weight percentage of rutile is obtained by subtracting the weight percentage of anatase from 100. According to the XRD results, from 60 to 80 °C, the anatase phase is formed. Increasing the synthesis temperature from 80 to 100 °C has led to partial conversion of anatase to rutile phase. To calculate the crystallite size (D), the Scherer equation where K is the Scherer constant (correction factor related to the sample shape and is equal to 0.9, λ beam wavelength, β peak width at half the maximum height, θ is the diffraction angle. Average crystallite size ( D ave ) can be resulted from Eq. (3) 23 : Table 1 shows that as synthesis temperature rise, crystallite size increments 24,25 , but once new crystalline phase is being formed, a decrease in average crystallite size is observed. From 60 to 70 °C, anatase single crystallites grow. The onset of rutile phase formation is 80 °C, the crystallite size gets a bit smaller. The crystal size reduced caused by the burst of nucleation was more palpable than that of the anatase crystal growth accelerated    Identification of ZnO nanoparticles. In order to compare the results of the microfluidic system with bulk, the molar ratio of ZnCl 2 to NaOH is kept constant at 0.6 with concentrations in the microfluidic system for the first and second streams are 30 and 50 mM, respectively (50 times dilution of the bulk systems). A microfluidic platform for synthesis comes along with more control over reaction, in the meantime, the reduction of chemicals consumption strongly prevents the occurrence of chemical accidents. Especially for highly acidic or basic circumstances. Synthesis time in room temperature spiral microfluidic system has been performed in 15 min V.S 2 h at 100 °C for the bulk approach. According to Fig. 7, the results of FTIR analysis associated with ZnO synthesis in the bulk and microfluidic system state the existence of identical peaks in the two samples. ZnO stretching vibrations are observed for both methods in the range of 400-668 cm −128 . In the range of 870-1000 cm −1 , there are Zn-OH peaks that are higher in number and intensity for nanostructures synthesized by the microfluidic method. In the range of 1200-1500 cm −1 , the C-OH bond, which can be formed as a result of the OH group due to washing with alcohol, indicates the bending vibration within -OH group in plain 29,30 . These peaks are seen more in the sample of the bulk method. Carboxylic group (C=O) was observed around 1700 cm −1 31,32 . A peak can be detected at 2300 cm −1 , which indicates the atmospheric absorption of CO 2 33 . In the range of 2900-2800 cm −1 , it shows the peaks related to asymmetric and symmetric stretches of -CH and -CH 2 groups. Peaks attributed to OH group stretching vibration have been identified at wave numbers higher than 3600 cm −1 . The presence of O-H stretches and hydrogen bonding by alcohol or water molecules can be found in peaks at 3400 or 3300 cm −1 34,35 .
The effect of precursors flowrate on ZnO size. The aim of this section is to investigate the morphology of ZnO nanoparticles synthesized in the spiral system at room temperature. Since the mixing in this type of microreactor is ultrafast, rapid nucleation is expected. A straightforward way is to tune the flow rate which influences the interface concentrations. Therein, the effect of the volumetric flow rate of ZnCl 2 and NaOH streams are considered in three cases (1) both: 25 µl/min (2) both: 50 µl/min (3) zinc chloride: 25 µl/min and sodium hydroxide flow: 50 µl/min.  Fig. 8 are provided in two magnifications of 500 and 1 µm. In the 1st case, where the volumetric flow of the two streams is low and equal, the residence time is high, leading to larger particles and a broader ZnO size distribution. In the 2nd case, when the flow rate doubles compared to the first case, the nanoparticle size is almost halved and the ZnO size distribution is somewhat reduced. In the 3rd case, where the ZnCl 2 flow rate is equal to 25 µl/min and the NaOH flow rate is equal to 50 µl/min, the particle size is intermediate. In addition, the narrowest size distribution is related to this case.  The results of FTIR analysis for bio-photo-catalyst components are shown in Fig. 9b. In the ZnO sample, ZnO tensile vibrations were determined with peaks of 701 and 669 cm -128 . In the TiO 2 /GOx/ZnO sample, a peak at 668 cm −1 could correspond to Ti-O-Ti and ZnO 42 . Two peaks in the range of 900-800 cm −1 refer to Zn-OH 29,30 . Peaks 939, 1101, 1159, 1206, 1226, 1297 cm −1 are related to Zn-OH and Ti-OH 42 . The presence of this bond indicates hydrolysis of the precursor 43 . For glucose oxidase, a peak at 1062 cm −1 was observed for the stretching vibrations of the C-O bond. In the meantime, this peak with a decreased intensity is detectable in the bio-photo-catalyst Peaks in the enzyme in the range of 1300-1400 cm −1 can also belong to the phenolic group of glucose oxidase 44 . In the bio-photo-catalyst spectrum, the peak in this region is also observed.
Two types of peaks were observed in all three samples. The presence of peaks in the range of 1400-1500 cm −1 may be related to the (CH)n bonds in the fatty acid or enzyme. Moreover, the C−H stretching modes of α-carbon and the aliphatic carbon chain of glutaraldehyde could be assigned near 2800 cm −1 41,45,46 . www.nature.com/scientificreports/ In the spectrum related to glucose oxidase enzyme, the presence of peaks related to amides of the first, second, and third types of peptide structure is also observed. At 1242 cm −1 and 1244, which are related to C-N and C-H tension and N-H torsion 47 . Receptor bonds of the first type were observed at 1600 cm -1 to 1700, which are related to the tensile vibration of C=O or CO peptide bonds in the protein structure, second amide bonds at 1500-1600 cm −1 , NH tension, and C-N tension of the peptide groups. Primary amines are commonly used to monitor structural changes in proteins, and this shows the biological activity of GOx 48,49 . These peaks coexist in the bio-photo-catalyst, however, compared to pure glucose oxidase, their intensity is diminished due to the bioconjugation. There is also a peak at 1743 cm −1 and 1699 which belongs to the carbonyl group 50 . In addition, GOx shows amide-related bonds at 3400-3440 cm −1 and amide B at about 2900 cm −1 , which originate from the Fermi resonance between the first peak of amide II and the N-H tensile vibration 51 . The peaks observed in the two samples ZnO and GOx in the range of 3300 cm −1 indicate the presence of surface water and O-H bond 42,46 . This peak confirms the presence of hydrophilic spots 31,32 . Optical study. UV-Vis diffuse reflectance absorption spectra (DRS) of TiO 2 , GOx, ZnO, TiO 2 /GOx, TiO 2 / GOx/ZnO in the range of 300-700 nm are shown in Fig. 10. Anatase TiO 2 , which is synthesized at 70 °C is utilized in this test. For TiO 2 /GOx, a shift of the absorption edge to the visible light region is witnessed. After 420.6 nm, the adsorption intensity ascribed to TiO 2 /GOx/ZnO is higher than bare TiO 2 .
The Tauc method is based on the assumption that the energy-dependent absorption coefficient α can be expressed by (αhν) 1/2 or (αhν) 2 = B (hν-E g ) where h is the Planck constant, ν is the photon's frequency, Eg is the band gap energy, and B is a constant 52 .The corresponding band-gap energy, calculated by using the Kubelka-Munk (KM) method and the plot of (αhν) 2 versus the photon energy (hν) for pure TiO 2 and ZnO are 3.20 and 3.33 eV, respectively. ZnO/TiO 2 heterojunction composite fibers has a bandgap of 2.9 eV 53 . Binary composite of TiO 2 /GOx has a bandgap of 2.43 eV, while for the TiO 2 /GOx/ZnO, bandgap is as low as 1.00 eV. Triple-heterojunction can improve the migration of photo-excited charge carries among different components to enhance photo-activity and charge separation 54 .
Bio-photo-catalytic degradation of amoxicillin. Amoxicillin synthetic wastewater with concentrations of 10-50 ppm is injected into the bio-photo-catalyst coated microchannel at a speed of 0.5 mm/min. The multiphase reaction has been performed for 1 h under UV light irradiation (0.25 W/cm 3 ). Figure 11a investigates the effect of synthesis temperature (70-120 °C) on the bio-photo-catalyst activity. In the whole range of synthesis temperature, can fully degrade amoxicillin up to 10 ppm. Figure 11a makes clear that at the same operational time, the lower concentration of amoxicillin and the lower synthesis time of TiO 2 , the higher the decomposition yield. The more pronounced adverse impact of increments in synthesis temperature rise can be observed in cases with great initial concentrations of amoxicillin. The effect of temperature can be interpreted by the SEM results of photo-catalysts synthesized in the microfluidic system. The photo-catalysts which are synthesized at 70 °C have the smallest diameter and length and size distribution. Figure 11b gives more details on the decrease of amoxicillin adsorption coefficient at different initial amoxicillin concentrations. Figure 11c, d study the effect of time on amoxicillin degradation over the bio-photo-catalysts with synthesis temperatures of TiO 2 in the range of (70-120 °C). According to the pseudo first-order reaction, the plot of ln(C 0 /C) against time should be linear. Kinetics of reaction reveal that apparent rate constants for samples (70-120 °C) are as 0.035, 0.027, 0.0206, 0.0206, 0.0152 min −1 , respectively. In the meantime, the behavior of the sample with 70 °C synthesis temperature defines with the highest yield and most rapidly degradation of amoxicillin.
For stability test, amoxicillin solution of 50 ppm illuminated by 0.25 W/cm 3 is fed up into the microreactor at 0.5 mm/min. In these series of experiments, the operational stability of the system in seven consecutive cycles is studied. The residence time was 1 h and no rising has been done between cycle intervals. In this work, only 8.1% alteration is seen in the amoxicillin degradation efficiency (Fig. 12). Similar results have been observed in where tetracycline is to be degraded in a microfluidic systems over ZnO/ZnS by 5% change in the efficiency after 5 cycles 55 . Microfluidic reactors provide uniform conditions of light propagation and flow distribution, which result in homogenous photoactivation of sites, regular interaction between active sites and pollutants, and eventually these systems allow higher stability compared to bulk reactors. Table 3 summarizes the published articles on the photo-catalytic amoxicillin degradation. In a recent work, the use of commercial titanium dioxide immobilized on the membrane to decompose amoxicillin (50 ppm) is reported in which, it is removed up to 80% after 500 min 56 . In another work, a hybrid nanostructure of TiO 2 /WO 3 with a calcination temperature of 700 °C has been utilized at a dose of 0.1 g/l to decompose 25 L of amoxicillin (100 ppm) and the intensity of UV solar radiation was set to a constant value, on a pilot scale. They achieved an efficiency of 64% 57 . TiO 2 as a slurry of titanium dioxide (anatase) at a reaction time and light intensity less than the previously stated articles, showed a 70% efficiency in the decomposition of amoxicillin, but its separation from the solution for reuse of photo-catalysts is more difficult 58 . The slurry sample had a yield reduction of about 13% under similar operating conditions, except that it was fixed on silica gel granules 59 . Exposure to visible light showed a significant effect of cobalt promoter on increasing the performance of TiO 2 in the decomposition of amoxicillin 60 , which enhanced the efficiency by as much as 70% increase compared to TiO 2. In another work, bismuth (as a promoter) and platinum were used to amplify TiO 2 in the photo-catalytic analysis of amoxicillin (10 ppm), which achieved an efficiency of 87% in 120 min under visible light 61 .
In the present work, a TiO 2-based bio-photo-catalyst coated capillary has been attained by modification of TiO 2 with glucose oxidase and oxide nanoparticles, and the complete decomposition of amoxicillin was irradiated with a 0.25 W/cm 3 UV-light source for 120 min at 50 ppm.
ZnO is dopant and it is not as influential as TiO 2 is. Therefore, its effect on the result is not that prominent. However, it is worth studying. Two findings are concluded from

Conclusion
In this work, the synthesis time in the microfluidic system is cut from hours to minutes (for TiO 2 each cycle is 25 min and for ZnO nanoparticles, it is 15 min). XRD results indicate that at a low temperature of 60 °C anatase formation is successfully attained. FTIR analysis indicate that ZnO nanoparticles which are synthesized in bulk and microfluidic systems are identical with respect to chemical bonds (ZnO stretching vibrations, Zn-OH peaks, C-OH bond, C=O groups, and O-H stretches). The biological activity of GOx enzyme is confirmed by Raman spectroscopy and FTIR tests that show the H 2 O 2 generation. The best temperature to synthesize TiO 2 is 70 °C; according to SEM results the nanoparticles are the finest and the size distribution is narrowest. Furthermore, the highest apparent rate constant of 0.035 min -1 and the highest yields of amoxicillin degradation (100% for 10 ppm, 93.9% for 30 ppm, and 87.8% for 50 ppm) ascribed to this sample. The findings of this work pave the way for upcoming researchers deciding to work on the development of new bio-photo-catalysts and their applications.

Data availability
All data generated or analyzed during this study are included in this published article.